Investigate the molecular mechanism that regulates male germline stem cell self-renewal or differentiation in DrosophilaAt the tip of the Drosophila testis (apex) is a germinal proliferation center, which contains the germline and somatic stem cells that maintain spermatogenesis. Each germline stem cell (GSC) is encysted by two somatic stem cells (SSCs). Both GSCs and SSCs anchor to a group of 12 nondividing somatic cells, called the "hub," through cell adhesion molecules. The hub defines the stem-cell niche by expressing the growth factor Unpaired (Upd), which activates the JAK/STAT pathway in GSCs to regulate stem-cell self-renewal. We have undertaken a number of genetic experiments in the last two years to further understand stem cell regulation and the role of the JAK/STAT signaling in this system. We have been focusing on:A-1. Identifying the signal (s) that regulates stem cell anchoring to the niche.In a genetic screen for mutations that interact with the JAK/STAT signal transduction pathway in regulating male GSC fates, we identified a small GTPase Rap guanine nucleotide exchange factor (Gef26) from our library of P-element mutations. We demonstrated that Rap-GEF/Rap signaling controls stem cell anchoring to the niche through regulating DE-cadherin-mediated cell adhesion (Wang et al., Dev. Cell 10:117-126, 2006). A-2. Identifying the downstream target of the JAK/STAT signal transduction pathway. Birt-Hogg-Dub (BHD) syndrome gene is a novel kidney tumor suppressor that was recently cloned by Dr. Berton Zbar's group in our laboratory. Dr. Zbar and I started collaboration on a project of studying the BHD gene's function in Drosophila. Through the use of RNAi to decrease expression of the Drosophila BHD homologue (DBHD), we have demonstrated that DBHD is required for male GSC maintenance in the fly testis. Further genetic interaction experiments suggest that DBHD regulates GSC maintenance downstream of the JAK/STAT and Dpp/TGF-? signal transduction pathways. These findings suggest that the BHD protein may regulate tumorigenesis through modulating stem cells in human (Manuscript submitted).A-3. Understanding how the GSCs and SSCs coordinate their self-renewal and differentiation. In many tissues, two or more types of stem cells share a niche, and how the stem cells coordinate their self-renewal and differentiation is poorly understood. In the Drosophila testis, germline stem cells (GSCs) and somatic stem cells (SSCs) contact each other and share a niche (the hub). The hub expresses a growth factor Unpaired (Upd) that activates the JAK/STAT pathway in GSCs to regulate the stem cell self-renewal. However, it is not clear how SSC cells are regulated and how SSCs and GSCs coordinate their self-renewal or differentiation. Through generating stat mutant SSC clones and selectively manipulating the JAK/STAT signaling level in either SSCs or GSCs, we demonstrated that the JAK/STAT signaling also regulates self-renewal of SSCs, but the JAK/STAT signaling in GSCs and in SSCs function differentially in coordinating self-renewal or differentiation of GSCs and SSCs. The somatic JAK/STAT signaling is essential for self-renewal and maintenance of both SSCs and GSCs; the germline JAK/STAT signaling is required for GSC self-renewal and maintenance but negatively regulates these functions in SSCs. These data suggest that a single signal from the niche regulates the self-renewal of both GSCs and SSCs; GSC-SSC interaction maintains their stability and coordinates their self-renewal or differentiation. (Manuscript submitted).A-4. Knocking out the mouse orthologs of Gef26. There are two mouse orthologs of Gef26, RapGEF1 and RapGEF2. There is a RapGEF1 mutant ES cell line available from Bay Genomics. We have purchased the ES cell line and are in the process of generating the RapGEF1 gene knockout mouse.